ABSTRACT

Neuroblastoma cell lines NB41A3 and SH-SY5Y were evaluated as an in vitro model system for studying organophosphorus (OP) chemical toxicity in central nervous system (CNS) cell lineages. Optimal culturing conditions, neuronal differentiation protocols and appropriate cholinergic gene expression were confirmed. The presence of muscarinic receptors and acetylcholinesterase activity was determined. Importantly, differential acetylcholinesterase inhibition by OP chemicals was demonstrated in live cells. This work has developed expertise in neuronal cell culture, confocal microscopy and enzyme activity assays that will provide the basis for an ongoing research programme. The neuroblastoma cell lines chosen can potentially be used as a model for investigating the toxicity of a range of CNS-acting chemicals of interest to the Department of Defence.

EXECUTIVE SUMMARY

The Human Protection and Performance Division (HPPD) of the Defence Science and Technology Organisation (DSTO) was tasked with developing a new research capability for investigating medical countermeasures to toxic organophosphorus (OP) chemicals, including chemical warfare nerve agents. HPPD does not have the facilities, personnel or funding for animal-based toxicology and pharmacology experimental programmes like those of our international Defence partners. It was considered, therefore, that the best avenue for novel research was to utilise cell culture models of the central nervous system (CNS). Such research effort will be complementary to the animal models utilised by our international Defence partners and will provide the opportunity to perform a basic scientific investigation of the precise biochemical mechanisms of nerve agent toxicity in the CNS where there remain many knowledge gaps. Such experiments can provide valuable information for subsequent animal-based medical countermeasure experiments.

The intention is to create an experimental platform and suite of techniques that can be applied to whatever chemical agents are of interest to the client. This platform will consist of cultured mammalian neurons and assays to investigate the cellular receptors and biochemical pathways that are targeted by chemical weapons. The utility of a given neuronal cell line for a given class of chemical warfare agent will need to be validated on a case-by-case basis. Cultured cells will need to express the required receptors and pathways that are applicable for the class of agent.

In order to initiate a nerve agent research programme, neuroblastoma cell lines were chosen and validated as a useful model system. Optimal culturing conditions for experimentation, neuronal differentiation protocols and appropriate gene expression were confirmed. The presence of muscarinic receptors and functional acetylcholinesterase was determined. Importantly, reliable assays for analysis of differential acetylcholinesterase inhibition by OP chemicals were generated using live neurons. This work has developed expertise in neuronal cell culture, confocal microscopy and enzyme activity assays that will provide the basis for an ongoing research programme. The neuroblastoma cell lines chosen can potentially be used as a model for investigating the toxicity of a range of CNS-acting chemicals of interest to the Department of Defence.